Biostimulants such as glycine betaine and kelp (Ascophyllum nodosum) extract are an emerging and innovative class of products that may mitigate the adverse efects of extreme heat. Results demonstrate genotype and biostimulants vary in their ability to mitigate heat stress over time. Results indicate biostimulants containing glycine betaine and kelp (Ascophyllum nodosum) extract enhance thermotolerance and may contribute to climate resiliency among commercial fruit crops exposed to heat stress.
Introduction
Climate change is a highly intricate challenge confronting humanity, with implications for global food security and the ability to supply essential vitamins and phytonutrients from fruit and vegetable crops. Global simulation models have predicted a 4-5 ℃ increase in atmospheric temperature by the year 2100, as well as a greater frequency of extreme heat events, which will reduce production if preemptive measures are not taken to enhance the resiliency of various horticultural crops, including fruit crops.
Reductions in plant production attributed to elevated temperatures and heat stress are associated with the disruption of several plant physiological processes, including leaf stomatal closure, chloroplast damage, and photosynthesis-related enzyme inactivation. Heat stress also disrupts evapotranspiration and slows multiple assimilation processes associated with photosynthesis. Reduced photosynthesis has been attributed to the thermal instability of Rubisco and the inhibition of the electron transport chain, as well as photosystem II (PSII), which limits photochemistry. This disruption is widely recognized to be due to heat-induced increases in thylakoid membrane fluidity and electron transport-dependent integrity of PSII. The inhibition of PSII activity after exposure to heat stress has been noted to result in reduced chlorophyll biosynthesis due to the inactivation of several enzymes. At the cellular level, heat stress may also cause protein and membrane denaturation and excessive production of harmful reactive oxygen species (ROS). Reduced carbon assimilation, lower photochemical efficiency, and excessive production of ROS often lead to poor crop growth and establishment.
Plants are sessile and can adjust morphologically, physiologically, and biochemically through acclimation processes, thereby minimizing the negative efects of heat stress. These processes can only be efective if they are activated and functional at the time of an extreme heat event. One potential way to prepare plants for heat stress treatment involves the use of biostimulants. Biostimulants are defined as agrochemical products formulated with mixtures of naturally occurring substances and/or microorganisms, which, when applied to plants in small quantities, can improve their mineral nutrition and tolerance to abiotic stresses such as heat and drought and improve crop yields while enhancing quality characteristics. Prior exposure to efective biostimulants may create a molecular memory within plants, priming them to be more tolerant or resistant to future abiotic or biotic stress. In general, there are nine categories of products classified as biostimulants: (i) humic substances; (ii) organic waste materials (e.g., sludge extracts, composts, and manures); (iii) beneficial chemical elements (Al, Co, Na, Se, and Si); (iv) chitin and chitosan derivatives; (v) seaweed extracts (brown, red, and green macroalgae); (vi) inorganic salts; (vii) anti-transpirants (kaolin and polyacrylamide); (viii) plant growth-promoting rhizobacteria (PGPR), arbuscular mycorrhizal fungi (AMF), and Trichorderma spp.; and (ix) free amino acids and nitrogen-containing substances (e.g., polyamines, peptides, and betaines).
Among the abovementioned biostimulants, seaweed extracts, beneficial chemical elements, and free amino acids and nitrogen-containing substances have garnered increasing attention within commercial horticultural industries for their potential ability to improve abiotic stress tolerance. For example, extracts from the brown seaweed Ascophyllum nodosum have been shown to improve overall plant productivity by stimulating chlorophyll biosynthesis and subsequently enhancing leaf color, promoting photosynthetic and antioxidant enzyme activities, increasing root and vegetative development, and enhancing flower and fruit development. The amino acids, proteins, vitamins, plant hormone precursors, growth promoters, trace elements, and other substances in these extracts may benefit plants under stress. Silica-rich biostimulants have also been noted to have beneficial efects on wheat (Triticum spp.) grown under abiotic stress. Previous studies revealed that silicon can alleviate ROS-induced stress in plant cells and tissues in several ways, including by attenuating the lipid peroxidation process of cell membranes, altering the levels of phytohormones, or activating the antioxidant defense system of drought-exposed sugarcane (Saccharum officinarum). Glycine betaine is an amino acid derivative associated with improved stress tolerance in a variety of crop species. Studies involving transgenic plants with increased glycine betaine production have shown that these plants have improved tolerance to abiotic stress. Glycine betaine has also been suggested to be involved in ROS scavenging, macromolecule (i.e., nucleic acids, proteins, lipids) protection under high NaCl concentrations, enhanced PSII repair, and functioning as a reservoir of carbon and nitrogen sources under abiotic stress.
Materials and methods
Measurements
Chlorophyll fluorescence
Leaf chlorophyll fluorescence, including minimal fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv), and maximum quantum efficiency of PSII (Fv/Fm), were collected weekly after each biostimulant application from four randomly selected pots per treatment. In each case, young, fully expanded leaves were measured using a Pocket PEA chlorophyll fluorometer (Hansatech Instruments Ltd., Norfolk, UK) with excitation light energy of 3500 μmol m−2 s−1. Readings were initiated at 1200 h and taken after 30 min of prior dark adaptation using light exclusion leaf clips.
Gas exchange
Gas-exchange measurements, including net CO2 assimilation (A), transpiration (E), and instantaneous water use efficiency (WUEi), were measured using a portable photosynthesis system (CIRAS-3, PP Systems; Amesbury, MA, USA). The automatic cuvette for the system was set at a leaf area of 4.5 cm2 and a CO2 concentration of 390 μmol mol−1. Again, data were collected weekly on young, fully expanded leaves from four randomly selected plants per treatment. The leaves were measured between 1000 and 1200 h and equilibrated for 2 min at a set temperature of 20 °C and a set photosynthetic photon flux density of 1200 μmol m−2 s−1. The weather was sunny on each date, and the plants were well watered.
Anthocyanins
Anthocyanins were extracted from 1-cm2 leaf disks that were collected from young, fully expanded leaves at 1 week after the final application of biostimulants. Four replicates per treatment were collected and quickly frozen in liquid nitrogen. Individual disks were placed into 2-mL Eppendorf tubes containing 1 mL of − 20 °C methanol/HCl/water (90:1:1, v: v: v) and held in the dark at 2 °C for 24 h. Anthocyanin content was then determined by measuring absorbance at 529 nm (A529) and 650 nm (A650) using a microplate spectrophotometer (Bio-Tek, Winooski, VT, USA). To correct for the effect of chlorophyll, we used the following empirically derived equation:
where AA is corrected anthocyanin absorbance. Total anthocyanin content was then calculated using the corrected absorbance and a molar absorbance coefficient for anthocyanin at 529 nm of 30,000 L mol−1 cm−1.
Biomass
Four plants per treatment were harvested destructively 1 week after the final application of biostimulants. Each plant was separated into shoots (leaves and stems) and roots (washed), oven-dried at 70 °C for 48 h, and weighed to determine the total dry biomass of the plants.
Results
Chlorophyll luorescence
Leaf chlorophyll fluorescence was significantly afected by interactions among genotype, biostimulant treatment, and the number of days after the biostimulant treatments were initiated during heat stress (P < 0.001). In the absence of biostimulants, the Fv/Fm ratio remained optimal in ‘Meeker’ (0.81-0.82) throughout the experiment (Fig. 1a). In contrast, the Fv/Fm decreased on day 7 in ORUS 4715-2, and both ORUS 4715-2 and WSU 2188 had lower Fv/Fm values than ‘Meeker’ on day 21. By day 28, these values had further declined to 0.72 in ORUS 4715-2 and 0.53 in WSU 2188. However, Fv/Fm did not decrease when Glycine Betaine was applied to all three genotypes (Fig. 1b) or when Kelp was applied to ‘Meeker’ and WSU 2188 (Fig. 1d). Furthermore, Fv/ Fm recovered at 28 days when Silicon was applied to WSU 2188 (Fig. 1c) but was afected by heat stress when Silicon or Kelp was applied to ORUS 4715-2 (Fig. 1c, d).
Gas exchange
A, E, and WUEi were significantly afected by interactions among genotype, biostimulant treatment, and the number of days after biostimulant treatments were initiated during heat stress (P < 0.001, < 0.001 and < 0.05, respectively). Without biostimulant application, A was consistently greater in ‘Meeker’ than in ORUS 4715-2 (Fig. 2a). Apart from day 28, WSU 2188 also had a greater A than did ORUS 4715-2. The results showed a similar pattern when the genotypes were treated with Glycine Betaine (Fig. 2b), Kelp (Fig. 2d), and, to some extent, Silicon (Fig. 2c). Notably, there was a general increase in the A of plants treated with Glycine Betaine from days 0 to 21 (A increased by 33%, 70%, and 66% in ‘Meeker’, WSU 2188, and ORUS 4715-2, respectively) and with Kelp from days 0 to 14 (A increased by 15%, 50%, and 31% in ‘Meeker’, WSU 2188, and ORUS 4715-2, respectively). The noted general increase inA was significant for ORUS 4715-2 and WSU 2188 on day 21 when both were treated with Glycine Betaine and Silicon (for ORUS 4715-2) relative to their controls (Fig. 3a). On day 28, both WSU 2188 and ‘Meeker’ exhibited greater A in response to treatment with Glycine Betaine (Fig. 3b) relative to their respective controls, and a similar response was noted for ORUS 4715-2 when it was treated with Silicon or Kelp.
Under control conditions, E was similar across the 28-day period for ‘Meeker’ and WSU 2188 and was consistently greater than that in ORUS 4715-2 on two or more dates throughout the study period (Fig. 2e). Similarly, E was generally greater in ‘Meeker’ and WSU 2188 than in ORUS 4715-2 when the plants were treated with Glycine Betaine (Fig. 2f). However, the E values were similar between WSU 2188 and ORUS 4715-2 on day 21, and both were lower than that of ‘Meeker’ on day 28. A generally linear increase in E from day 0 to 28 was also observed when ‘Meeker’ and WSU 2188 were treated with Glycine Betaine. Except for day 0, E was similar among the genotypes treated with Silicon (Fig. 2g). Higher rates of E were observed on days 0, 14, and 28 for ‘Meeker’ in comparison to ORUS 4715-2 after treatment with Kelp (Fig. 2h). Treating both ‘Meeker’ and WSU 2188 with Glycine Betaine and Kelp increased E on day 28 relative to their controls, whereas Kelp only increased E in ORUS 4715-2 (Fig. 3c).
Without biostimulants and on day 0, the WUEi was similar between ORUS 4715-2 and ‘Meeker’ and was greater than that in WSU 2188 (Fig. 2i). However, on day 7, the WUEi of ORUS 4715-2 plummeted and was significantly lower than that of ‘Meeker’ and WSU 2188 on day 14. Overall, the ‘Meeker’ control maintained a relatively high WUEi throughout the 28-day treatment period. The application of Glycine Betaine increased WUEi in ‘Meeker’ on day 28 but did not lead to improvements in WSU 2188 or ORUS 4715-2 (Fig. 2j). However, a greater WUEi was observed on day 28 when ORUS 4715-2 was treated with Silicon (Fig. 2k). Kelp resulted in greater WUEi in ‘Meeker’ and WSU 2188 than in ORUS 4715-2 on day 21 (Fig. 2l).
Anthocyanins
Anthocyanins in the leaves were significantly afected by the interaction between genotype and biostimulants (Table 1). The anthocyanin content was low and similar among the genotypes in the control treatment but was 3- to 11-fold greater with Glycine Betaine or Kelp than with either Silicon or the control in ‘Meeker’ and WSU 2188.
| Biostimulant | Anthocyanins (μmol cm–2) | ||
| Meeker | WSU 2188 | ORUS 4715-2 | |
| Control (untreated) | 0.3 ± 0.1c | 0.3 ± 0.1c | 0.2 ± 0.1c |
| Glycine Betaine | 3.4 ± 1.0a | 1.8 ± 0.4ab | 0.9 ± 0.2bc |
| Silicon | 0.6 ± 0.1bc | 0.6 ± 0.1bc | 0.4 ± 0.3bc |
| Kelp | 3.1 ± 0.1a | 2.3 ± 0.7ab | 0.7 ± 0.2bc |
| F value | |||
| Genotype (G) | 9.6*** | ||
| Biostimulant (B) | 15.9*** | ||
| G x B | 2.8* |
Biomass
Shoot, root, and total dry biomass were significantly affected by genotype, whereas only total biomass was impacted by biostimulant treatment (Table 2). On average, ORUS 4715-2 produced less shoot and total biomass than did the other two genotypes and less root biomass than did WSU 2188. However, regardless of genotype, plants treated with FRUIT ARMOR produced more total biomass than did the control plants treated with water only.
| Biostimulant | Dry biomass (g/plant) | ||
| Shoot | Root | Total | |
| Genotype | |||
| Meeker | 9.6 ± 0.1a | 2.5 ± 0.1ab | 12.1 ± 0.9a |
| WSU 2188 | 8.7 ± 0.1a | 2.9 ± 0.1a | 11.6 ± 0.6a |
| ORUS 4715–2 | 5.3 ± 0.1b | 1.6 ± 0.1b | 6.8 ± 0.5b |
| Biostimulant | |||
| Control (untreated) | 7.1 ± 1.0 | 2.0 ± 0.3 | 9.0 ± 1.1b |
| Glycine Betaine | 9.2 ± 0.8 | 2.9 ± 0.4 | 11.8 ± 1.0a |
| Silicon | 8.4 ± 0.9 | 2.1 ± 0.4 | 10.5 ± 1.1ab |
| Kelp | 7.3 ± 0.8 | 2.1 ± 0.4 | 9.3 ± 0.9ab |
| F value | |||
| Genotype (G) | 15.7* | 8.8* | 21.1* |
| Biostimulant (B) | 2.4 | 2.3 | 3.6* |
| G x B | 1.7 | 0.9 | 1.4 |
Discussion
In the present study, we found diferences in the ability of three biostimulant products, Glycine Betaine, Silicon, and Kelp, to induce plant tolerance to heat stress. To the best of our knowledge, this is the first study to test these products in raspberry. Five applications of Glycine Betaine over a 28-day period enhanced the Fv/ Fm and gas exchange and increased the anthocyanin content and total biomass of the plants. Kelp also improved physiological parameters, including Fv/Fm, gas exchange, and anthocyanin content. Past studies have indicated that both foliar and soil applications of seaweed extract and glycine betaine are beneficial for other crops, including tomato (Solanum lypopersicum) and barley (Hordeum vulgare), and can activate growth, adjust metabolic processes, alleviate heat stress, and improve plant fitness.
Observed in WSU 2188 and ORUS 4715-2 were likely caused by structural alterations in the PSII complex that hindered energy transfer from the light harvesting antenna to the PSII reaction centers, the physical separation between PSII reaction centers and the peripheral antennae, and/or the inhibition of the photosynthetic electron transport chain.
Interestingly, an increase in the Fv/Fm ofWSU 2188 and ORUS 4715-2 occurred when both genotypes were treated with Glycine Betaine. The active ingredient of Glycine Betaine is glycine betaine, an amine with an ampholytic nature that has naturally been associated with abiotic stress tolerance in various organisms. During abiotic stress, the synthesis of proteins involved in PSII repair is afected, leading to photoinhibition. However, glycine betaine appears to antagonize the inhibition of protein biosynthesis and thus enhances the repair and maintenance of PSII structural integrity, leading to increased stress tolerance. Exogenous application of glycine betaine protected PSII in heat-stressed barley. It is demonstrated that the overaccumulation of glycine betaine by the transformation of a glycine betaine synthesis gene into plants can protect the photosynthetic apparatus from heat damage. Exogenous application of glycine betaine seems to mediate heat stress and may have led to the maintenance and improvement of photochemical efficiency after treatment with Glycine Betaine.
The application of Kelp also improved the Fv/Fm ofWSU 2188 on days 21 and 28 after application of biostimulant treatments to levels comparable to the established Fv/Fm values for non-stressed plants. Although still lower than the established values for non-stressed plants, an improvement in the Fv/Fm ofORUS 4715-2 was also observed on these days relative to that of the control treatment. Genes encoding subunits of PSII involved in chlorophyll biosynthesis (e.g., protochlorophyllide oxidoreductase) and the Calvin-Benson cycle (e.g., Rubisco) were not afected by oxidative stress in Arabidopsis thaliana plants treated with Ascophyllum nodosum. The upregulation of the expression of some of these genes potentially enhances the photosynthetic efficiency of biostimulant-treated plants and provides protection against stress. In their study using tomatoes, Carmody et al. suggested that Ascophyllum nodosum biostimulant treatment has the potential to increase the energy available for PSII photochemistry to increase crop yield under mild and chronic heat stress.
Consistent with Fv/Fm, the application of Glycine Betaine also improved the photosynthetic performance of each genotype. From days 0 to 21, A increased by 33% in ‘Meeker’, 70% in WSU 2188, and 66% in ORUS 4715-2 when Glycine Betaine was applied. Chloroplast-produced glycine betaine has been noted to protect the photosynthetic apparatus, shielding enzymes and lipids required to maintain optimal linear electron transport flow through thylakoid membranes. The application of glycine betaine to apple (Malus domestica) resulted in enhanced photosynthetic performance under heat, drought, and a combination of both stresses. This ability to sustain leaf gas exchange under heat stress has been directly correlated with thermotolerance in several plant species. Hence, the exogenous application of Glycine Betaine might have played a role in maintaining stomatal conductance and E in ‘Meeker’ and WSU 2188, in contrast to the decline observed on day 28 in heat-stressed control plants across the three genotypes.
Consistent with the other physiological results, Glycine Betaine and Kelp treated ‘Meeker’ and WSU 2188 plants accumulated more anthocyanins relative to the control treatments. There is evidence that anthocyanins protect the photosynthetic apparatus from photoinhibition by absorption of green light, thereby reducing excess excitation energy. It is reported that anthocyanins might provide photoprotection by enhancing their antioxidative capabilities under high temperature stress, and it is noted that the ROS scavenging role of anthocyanins helps maintain the photosynthetic capacity to aid plant survival. This potential enhanced antioxidative capability under temperature stress in plants treated with Glycine Betaine and Kelp could have reduced ROS damage to the thylakoid membrane and photosynthetic apparatus. Biostimulants derived from amino acids, enhanced the activity of phenylalanine ammonia-lyase. The enzyme serves as an initial step in the phenylpropanoid pathway, which is the starting point for secondary metabolic pathways of various phenolic compounds, including anthocyanins.
In conclusion, ‘Meeker’ and WSU 2188 maintained higher Fv/Fm and gas-exchange rates under control and biostimulant treatments, a result that manifests in improved heat tolerance in the raspberry genotypes. Similarly, the application of Glycine Betaine resulted in greater accumulation of anthocyanins in ‘Meeker’ and WSU 2188 plants, as well as improved Fv/Fm, enhanced gas exchange, and greater total biomass. FRUIT ARMOUR also improved the Fv/Fm of the more heat-sensitive ORUS 4715-2 genotype. Together, these factors indicate that glycine betaine contributes to enhanced repair and maintenance of PSII structural integrity and improved photosynthetic performance and antioxidative capabilities. This, in turn, may have ledto the greater total biomass of the raspberry plants treated with Glycine Betaine relative to that of the untreated controls. Kelp also improved Fv/Fm and anthocyanin accumulation of WSU 2188, as well as the A of ORUS 4715-2. This efect of Kelp was attributed to the contribution of Ascophyllum nodosum to increased energy availability for PSII photochemistry and the upregulation of genes encoding subunits of PSII involved in chlorophyll biosynthesis. The contributions of both biostimulant products to improved physiological and growth performance under heat stress suggest their potential role in enhancing the thermotolerance of raspberry and other fruit crops exposed to heat stress. Therefore, commercial fruit crops threatened by heat stress may benefit from the application(s) of Glycine Betaine, Kelp, or similar products containing these active ingredients. However, further research is required to identify ideal application rates, times (growth stage), and methods under field conditions involving mature plants producing fruit. Cost‒benefit analyses are also warranted to understand the economic implications of these inputs in commercial agricultural systems.
Conclusion
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